Therapy for colorectal and small intestine cancers

This application is a U.S. national stage filing under 35 U.S.C. § 371 from International Application No. PCT/US2020/024012, filed on Mar. 20,2020, and published as WO2020/191356 A1 on Sep. 24, 2020, which claims benefit of priority to the filing date of U.S. Provisional Application Ser. No. 62/821,546, filed Mar. 21, 2019, the contents of which are specifically incorporated by reference herein in their entirety.

This invention was made with government support under R35 CA197588, HD067244, and R01 NS093872 awarded by the National Institutes of Health and under 1K22CA216036 and K08 CA230318 awarded by the National Cancer institute. The government has certain rights in the invention.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 4, 2022, is named 2259548.txt and is 131,072 bytes in size.

Colorectal cancer (CRC) is the third-leading cancer type in the world and contributes to 7.9% of the world cancer-related deaths in 2000. In Japan, CRC is the leading cause of death and the number of the patients has been increasing every year. In the United States, there is a rising incidence of CRC in young adults. The development of an agent and therapeutic method which are highly effective and safe is strongly desired.

The five-year survival rate of colorectal cancer is relatively high with stage I and stage II because the cancer lesions can be removed almost completely by surgical operation. However, this rate is significantly reduced with advanced cancer (stage III and stage IV).

Methods are described herein that include (a) reducing or eliminating sucrose, fructose, glycine, serine, or a combination thereof from a subject's diet; (b) administering a GLUT5 inhibitor; (c) administering a ketohexokinase (KHK) inhibitor; (d) administering a fatty acid synthase (FASN) inhibitor; (e) administering a phosphoinositide 3 (PI3) kinase inhibitor, or (4) a combination two or more thereof to inhibit the onset of colorectal or small intestine cancer or to reduce colorectal or small intestine tumor growth in the subject.

As shown herein, high-fructose corn syrup enhances intestinal tumor growth and the incidence of high-grade tumors. Such cancer/tumor growth can be inhibited or prevented by genetic deletion of ketohexokinase (KHK), the major enzyme that initiates fructose metabolism, or fatty acid synthase (FASN). Methods and compositions are described herein for dietary changes and therapeutic inhibition of (1) fructose transport (via GLUT5), (2) metabolism (KHK), (3) fatty acid synthesis (FASN), (4) phosphoinositide 3-kinases, or (5) a combination thereof to inhibit and/or prevent tumor growth. Such methods can also include modifications of diet including to reduce or eliminate consumption of certain types of amino acids, sugars and/or carbohydrates. Also, as illustrated herein, the KHK-derived metabolite, fructose 1-phosphate (HP), allosterically inhibits pyruvate kinase M2 (PKM2). This inactivation can accelerate tumor growth. Hence, small molecules that activate PKM2 (e.g. TEPP-46) may also inhibit and/or prevent intestinal tumor growth.

Methods and compositions are described herein that can be used to inhibit or delay the onset of certain types of cancer, including colorectal cancer and cancers of the small intestine. Colorectal cancer is one of cancer species which occurs in the large intestine (cecum, colon and rectum). Cancers that occur in the anal canal are also included in the definition of colorectal cancer, as well as intestinal polyps or adenomas that may eventually turn into cancer. In general, the colorectal cancer is divided into cecum cancer, colon cancer and rectum cancer. In some cases, the patient or subject that is treated has an adenomatous polyposis coli (APC) genetic mutation. The methods and compositions described herein can be used to inhibit or treat patients with APC mutations and/or any of these cancer types.

The methods and compositions described herein can inhibit (1) fructose transport (e.g., via GLUT5), (2) metabolism (e.g., via ketohexokinase, KHK), (3) fatty acid synthesis (e.g., via FASN), (4) phosphoinositide 3-kinase (PI3K), or (5) a combination thereof to inhibit and/or prevent tumor growth. The methods can include modifications of diet such as reducing or eliminating fructose from the diet, reducing or eliminating sugars from the diet, reducing or eliminating certain amino acids from the diet, reducing or eliminating carbohydrates from the diet, or combinations thereof. For example, the methods can include use of a ketogenic diet, a diet with a low glycemic index, or a sugar-free diet.

Diet

The methods described herein can include reducing or eliminating certain sugars, carbohydrates, amino acids, and combinations thereof.

Sugars and carbohydrates that include high amounts of sucrose, glucose, and especially fructose, can be reduced or eliminated from the diet to reduce the incidence and progression of cancers such as colorectal cancers and cancers of the small intestine. In general, to reduce the incidence and progression of cancers such as colorectal cancers and cancers of the small intestine the diet should have less than about 25 grains of sugar per day, or less than about 20 grains of sugar per day, or less than about 15 grams of sugar per day, or less than about 10 grams of sugar per day, or less than about 5 grams of sugar per day.

Some types of sugar are more problematic than other types. High fructose corn syrup consists of glucose and fructose in a 45:55 ratio and the amounts ingested should be reduced because it contains so much fructose. Honey and tapioca syrup also contain significant amounts of fructose. High levels of fructose are problematic because such levels accelerate glycolysis and de novo lipogenesis that support tumor growth.

Examples of foods that contain fructose and should be avoided include high fructose corn syrup, sugar-sweetened beverages (SSBs, which are primarily sweetened with high-fructose corn syrup), honey, tapioca syrup, candy, sweetened yogurt, salad dressings sweetened with sugars or high fructose corn syrup, frozen or boxed dinners dressings sweetened with sugars or high fructose corn syrup, frozen pizzas sweetened with sugars or high fructose corn syrup, breads dressings sweetened with sugars or high fructose corn syrup, canned fruit sweetened with sugars or high fructose corn syrup, fruit juices, and granola bars sweetened with sugars or high fructose corn syrup. However, some types of fruits and vegetables also contain significant amounts of fructose including apples, grapes, watermelon, asparagus, peas, and zucchini, which should also be avoided in some cases.

In some cases, the subject's diet can be a fructose-free diet, or a diet that is substantially reduced in fructose, combined with a KHK inhibitor (e.g., any of these described herein). One example of a KHK inhibitor that can be used is a PF-06835919 inhibitor.

Foods that include high amounts of the amino acid glycine can be reduced or eliminated from the diet to reduce the incidence and progression of cancers such as colorectal cancers and cancers of the small intestine. In general, a diet to reduce the incidence and progression of cancers can include less than 10 grams per day, or less than 7 grams per day, or less than 5 grams per clay, or less than 4 grams per day, or less than 3 grains per day, or less than 2 grams per day, or less than 1 gram per day of glycine.

Glycine is abundant in cartilage, collagen, bones, tendons, and gelatin. Examples of foods containing significant amounts of glycine that can be avoided to reduce the incidence and progression of cancers include gelatin, pork skins, pork ears, pork feet, meat-by-products, jellied beef luncheon meat, chicken breast, corned beef, ostrich, crustaceans (crab, Alaska king crab, mollusks, lobsters), etc. The following website provides a listing of foods high in glycine nutritiondata.self.com/foods-000094000000000000000.html. The following website provides listings of low glycine foods: eatthismuch.com/food/browse/low-glycine-foods/?q=&type=food&page=3&order_by=glycine&show_nutrient=glycine.

Foods that include high amounts of the amino acid serine can be reduced or eliminated from the diet to reduce the incidence and progression of cancers such as colorectal cancers and cancers of the small intestine. In general, a diet to reduce the incidence and progression of cancers can include less than 10 grams per day, or less than 7 grams per day, or less than 5 grams per day, or less than 4 grams per day, or less than 3 grams per day, or less than 2 grains per day, or less than 1 gram per day of serine.

Foods containing significant amounts of serine include fish (salmon, hake, monkfish, cod, and fish broth), milk, eggs, cheeses, beans, carob seeds, soy (tofu, tempeh, soymilk), peanuts, asparagus, yogurt, and lentils. The following website provides a listing of foods high in serine: nutritiondata.self.com/foods-000096000000000000000.html.

In some cases, the diet can be a serine/glycine depleted diet together with one or more phosphoglycerate dehydrogenase (PHGDH) inhibitors. Such PHGDH inhibitors include any of the following: PH719, NCT-502, NCT-503, TDI-8077, TDI-6570, CBR-5884, CBR-5807, CBR-6936, CBR-9480, PKUMDL-WQ-2201, PKUMDL-WQ-2101, alpha-ketothiomide inhibitor, AZ compound, Raze compound.

Some examples of PHGDH inhibitors are shown below.

In some cases, the diet can be a ketogenic dict that is highly palatable and easy to consume. A ketogenic diet involves ingestion of more calories from fat and less from carbohydrates. Hence a ketogenic diet is classified as a low, or very low carbohydrate diet. Sugars are generally eliminated or significantly reduced from a ketogenic diet. For example, a subject's ketogenic diet can involve ingestion of less than 30 grams carbohydrate per day, less than 20 grains carbohydrates, less than 15 grams carbohydrate per day, less than 10 grams carbohydrates, less than 7 grams carbohydrate per day, less than 5 grams carbohydrates, or less than 3 grams of carbohydrates per day. In some cases, the Atkins diet (an example of a high fat and high protein diet) can be used as a ketogenic diet.

In some cases, such a ketogenic diet can involve ingestion of a 3:1 ratio of ketogenic-to-antiketogenic macromolecules, which results in approximately 85% fat, 12% protein, and 3% carbohydrates. There is a diverse mixture of fats. For example, the fats can include those from plants, nuts, and animal products. The diet can be actively managed by dieticians who interact with patients on the diet on a weekly basis. Such a diet can obtain up to 80% compliance, up to 90% compliance, up to 95% compliance, up to 96% compliance, up to 98% compliance, up to 99% compliance, or even up to 100% compliance. For example, 100% compliance over 4 weeks was achieved in an ongoing pilot study in women with endometrial cancer.

In some embodiments, the ketogenic diet includes at most 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% protein, with the remainder of the diet made up of fat, fiber, ash, and carbohydrates. In some embodiments, the ketogenic diet includes at most 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% carbohydrates, with the remainder of the diet made up of fat, fiber, ash, and protein. In some embodiments, the ketogenic diet includes fat measured in grams and carbohydrates and proteins collectively measured in grams in a ratio of 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, or 4.5 to one (1) of fat to carbohydrate/protein. A comparison of a ketogenic diet with a normal diet is shown below as Table 1.

The methods described herein can include such a ketogenic diet with administration of PI3 kinase, GLUT5, KHK, FASN, PHGDH inhibitors, including any of those described herein.

In some cases, for any of the methods disclosed herein, “administration” includes providing one or more of a PI3 kinase inhibitor, GLUT5 inhibitor, KHK inhibitor, FASN inhibitor, PHGDH inhibitor, and/or ketogenic diet to the subject, e.g., to be ingested or administered at the same or a later time, or providing a prescription for one or more of a PI3 kinase inhibitor, GLUT5 inhibitor, KHK inhibitor, FASN inhibitor, PHGDH inhibitor, and/or ketogenic diet to the subject. In certain embodiments, “administration” of the ketogenic diet comprises instructing the subject to follow a ketogenic diet.

GLUT5

GLUT5 is a fructose-transporter, and a member of the facilitative glucose transporter (GLUT, SLC2) family. One example of aGLUT5 protein sequence is shown below as SEQ ID NO:1 (NCBI accession no. NP_001315548.1).

An example of a cDNA that encodes the GLUT5 protein with SEQ ID NO:1 is shown below as SEQ ID NO:2 (with NCBI accession no. NM_001328619.1).

Another example of aGLUT5 protein sequence is shown below as SEQ ID NO:3 (NCBI accession no. AAA52570.1).

An example of a cDNA that encodes the GLUT5 protein with SEQ ID NO:3 is shown below as SEQ ID NO:4 (with NCBI accession no. M55531.1).

Subjects can express a GLUT5 enzyme that can have one or more amino acid differences compared to the sequences described herein. For example, subjects can express a GLUT5 enzyme at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% amino acid sequence identity or similarity with the GLUT5 amino acid sequences described herein. Similarly, subjects can express GLUT5 RNA with one or more nucleotide differences compared to the GLUT5 nucleic acids described herein. For example, subjects can express a GLUT5 RNA at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% amino acid sequence identity or similarity with the GLUT5 nucleic acid sequences described herein.

As described herein, inhibition of GLUT5 can inhibit cancer, including colorectal cancer and cancers of the small intestine. One example of an inhibitor of GLUT5 is N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA), with the following structure. See WO2016201214A1.

Other examples of GLUT5 inhibitors include N-(2,5-dimethoxybenzyl)-N-[4-(methylsulfonyl)-2-nitrophenyl]amine; N-(3,4-dimethoxyphenyl)-4-(methylsulfonyl)-2-nitroaniline; N-[1-(3-fluoro-4-methoxypheny 1) ethyl]-4-(methylsulfonyl)-2-nitroaniline; N-[1-(1,3-benzodioxol-5-yl) ethyl]-4-(methylsulfonyl)-2-nitroaniline; and N-(3,5-dimethoxyphenyl)-4-(methylsulfonyl)-2-nitroaniline. See WO2016201214A1. Structures of some GLUT5 inhibitors are shown below.

Additional inhibitors of GLUT5 are provided in JP 2015-205827 A.

Ketohexokinase (KHK)

Ketohexokinase (KHK) is a fructokinase that catalyzes the phosphorylation of fructose to fructose-1-phosphate (F1P) in the first step in fructolysis. The C isoform of fructokinase is the predominant form of the enzyme in the liver, kidney and intestine, whereas the fructokinase A splice variant is expressed in most tumors and throughout the body. There are at least 12 other KHK isoforms expressed in humans.

One example of an amino acid sequence for aisoform C of fructokinase (KHK) is shown below as SEQ ID NO:5 (NCBI accession no. NP_006479.1).

An example of a cDNA that encodes the KHK protein with SEQ ID NO:5 is shown below as SEQ ID NO:6 (with NCBI accession no. NM_006488.3).

Another example of aKUM protein sequence is shown below as 110 SEQ ID NO:7.